Preparation of blood fraction for use in rh testing procedures



United States Patent invention relates to a process for'the fractionation of'mammalian serum and relates particularly to a process for preparing a serum fraction containing albumin in a major amount and globulin in a minor amount, said serumfraction being suitable for use inldemonst'ratingthe presence in blood of an Rhantibody known variously as incomplete antibody,v blocking antibody, albumin agglutinin, and agglutinoid.

In 1939, Levine and Stetson, J. A. M. A., 1131126, 1939.

reported a transfusion reactionin a-woman who delivered a macerated'fetus and whose serum contained agglutinins. In 1940,. Landsteiner and Wiener, Proc. Exper. Biol. and Med. 43':223, 1940, reported the preparation of an antiserum againstthe red cells of the rhesus monkey and.

found that this anti-Rhesus serum, when properly absorbed, agglutinatedthe blood of approximately 85 per cent of the members of the white race that were tested. They labelled. the antigen in the human bloods that reacted .witlithe anti-Rhesus serumthe Rh factor. Human bl'oodsf containing the Rb factor weretermed-Rh-positive. Itwas. shown that. Levine and Stetson, who reported the earlier transfusion. reaction, were dealing with a'case of, Rh immunization and thatthe unnamed'property was the property later named the Rb factor by Landsteiner and .Wiener- Levine, Katzin and Bumham, I. A. M. A., 1.16:825, 1941, reported that intragroup incompatibility copldbe produced. by immunization of an Rh-negative mother'by an. Rh-positive fetus. These investigators demonstrated at the same time that Rh immunization explained themajority of cases of erythroblastosis fetalis.

It was at first-thought that the R11 types couldbe explained. on the basis 0f thepresenceor absence of the Rlyfactor, a single positive :factor. Laterit was discovered. that the. problem was. much more complex and atithe present. time at leasteight Rh subtypes. are demone strable'.. The anti-Rhv agglutininslare not foundnormally in. the blood of. any individual. Antibodies. that, agglutinate 'Rh-po'sitivel cellsmay develop in the serum of some...Rh-negative individuals undercertain conditions. Anti-Rh-agglutirnnswhich appear in the bloodof certain.

Rh negative individuals. as .a result .of immunizationby transfusion ofRh-positivebloodior by bearingRh-positive children, are demonstrable in the serum or plasma of tbe'immuni'zed individual and are generally divided into two types;, the saline agglutinins (complete antibodies) and albumin agglutinins (incomplete antibodies). Anti- Rh' a'gglutininsin-general.are similar to otherisohemagglutinins except that they are more active at 37 C. than at lower temperatures. The anti-Rh agglutinins act-very well-when the serum istest'ed against 211% or 2% suspension of Rh positive cells'in 0.85% sodium chloride. It was later discovered that there were'ant'ibodies other thanthe saline-active; anti-Rh agglutinins responsible forfiminunization. It was noted that" a large proportion of the Rh negativ'e women'who bore erythroblastotic children did noti show'anti-Rh agglutinins'in their serum when the serum was tested against a 'I'%or2'%"suspen- 2 sion of Rh-positive cells in 0.85% sodium chloride; It was demonstrated that these women were also immunized against the Rhfactor even though Rh antibodies were not demonstrated when the serum was tested against Rhpositive cellssuspended in salt solution. I

It was later demonstrated that the serumof'itho'se' women had the capacity to prevent the action of known anti-Rh agglutinins against Rh-positive cells suspended in salt solution and it was assumed that their serum possessed antibodies capable of-nullifying the efiect of ordinary anti-Rh agglutinins on Rh-positivecells suspended in saline. This antibody has beencalled incom plete antibody, blocking. antibody, albumin agglutinin, and agglutinoid.

A test for demonstrating the presence of the-incomplete antibody was developed by Diamond and Abelson,- J. Lab. and Clin. Med, 30:204, 1945, which demonstrated this antibody produced agglutination of Rh-posi-v tive cells; In the test, one drop of the serum-containing the incomplete antibody was mixed with two drops of whole oxalatedor citrated Rh-positive blood and spreadthinly on awarm slide, and this resulted in. clumping: of the Rh-positive cells. The same results wereobtained and could be demonstrated by testing the serum contain: ing. the incomplete antibody against a 50% suspension of Rh-pOSifiVe cells suspended inserum. It was'orig-inally thought that the heavy cell suspension'wasa factor which permitted such a serum. to clump Rh-positive'cells; but it was later concluded that the serum or plasma in;.which the cells were suspended was the determining factor. It was still later shown-that the same effect is possiblefwhen incomplete antibody is tested against a 2% suspension of Rh-positive cells suspended in' a 20% to 30% solution of human or bovine albumin, acacia, globin and many other macromolecular substances. agglutination by incomplete antibodies was later modified and in this modification; the serum and Rh-positive cells in saline are incubated for one hour at 37 C and centrifuged. The supernatant saline is removed and serum or other: macromolecular substance added followed by re' suspension of the cells. The mixture is again centrifuged and observed for clumping. In this test, the Rh-positive cells in saline absorb the incomplete antibody'buf do' noti inthe efiiciency of different preparationsof albumin sug gested that the activating protein was not albumin but some contaminating protein associated with it. Kekw'ick and MacKay, Proc. First International Congress of Biochemistry, 1949, report that solutions of betaand'gammaglobulin preparedby the ether fractionation of human plasma were inactive but that an active fraction could be prepared from the supernatant left after their precipi'-' t'ation which was soluble in 0.9% saline at pH 5.0. "This soluble fraction consisted of albumin 38%; alpha-globu'-.'

lin, 33.4%; beta-globulin, 19.7%; and gamma-globulin,

8.9%. Albumin, separated electrophoretically from this" preparation and concentrated by freeze-drying, showed" no activity, but at a comparable protein concentration, the globulins were highly active- McCulloch concluded from these reported results that since the'albumin was. consistently negative, and no activity could be found iii beta-and gamma-globulin; the activity appeared to be a's-' sociatedwith alpha-globulin. Thisauthor suggested that. alpha-globulin is responsible for the demonstrationof:

incomplete Rh antibodies in the Diamond test.

F'FL v A I Pateiited' Sept. 4,1956

' within the pI-I' ranges specified above.

It is an object of this invention to prepare a serum fraction suitable for use in demonstrating incomplete antibodies in a method described by Diamond.

It is another object of this invention to prepare a serum fraction suitable for use in demonstrating incomplete Rh an'tibodies by suspending the reacting cells in a concentration of the serum fraction. a -It" is still another object of this invention to prepare a serum fraction, suitable for use in the method of demonstrating incomplete antibodies of Diamond in which thereacting cells are suspended in concentrated albumin solution, which fraction contains albumin and alphaglobulins. It has now been discovered that a serum fraction containing albumin and alpha-globulins which is suitable for use in the Diamond test may be produced from mammalian blood'serum by, a selective precipitation process in which, by a first precipitation step, globulins other than alpha-globulin are denatured, and precipitated from serum containing an alkali metal salt of an organic acid dissolved therein "while alpha-globulin and albumin remain in solution in an undenatured condition; and a second precipitation step, in which alpha-globulin and albumin are precipitated by addition of a lower aliphatic alcohol to the solution after removal of previously precipitated denatured globulins other than alpha-globulins.

In the process of this invention, an alkali metal salt of a fatty acid having from four to ten carbon atoms,

test and at a molarity substantially above this range, alpha-globulin's areprecipitated. Salts of acids such as butyric, caproic, and hexanoic have been found particular The pH of the solution is adjusted to ap ly suitable. proximately. 6.8 to 8.2; the preferred pH range-is 7.0 to 8.0. The temperature of the solution is raised and maintained substantially above room temperature for a short period of time, preferably 25 to 35 minutes; the preferred temperature to which the solution is brought is 70 to I 75. Following the heating period, the solution is cooled to a temperature approximating room temperature. The presence. of the alkali metal salt of the acid in the serum prevents heat inactivation of alpha-globulins and albumin and the region of greatest stability of these two substances in the presence of the alkali metal salt of the acid is Globnlins other than alpha-globulins are denatured by the heat treatment. The pH of the cooled solutionis' adjusted to 5.55 to 5.60. At a pH within this range denatured beta-globulins precipitate and are removed by anysuitable means such as centrifugation. If the pH is adjusted to lower than 5.55, some alpha-globulinsare precipitated and if the pH is adjusted to' higher than 5.60, the denatured globulins are not completely precipitated. The pH of the solution after removal of the beta-globulins is adjusted to 3.0 to 5.5;

the preferred pH range is 3.3 to 4.8. Alpha-globulins are J precipitated at a pH within this range and the isoelectric point of the albumin is also within this range. If the pH is below "3.0, albumin is destroyed and if the pH is above 5.5, alpha-globulins are not precipitated. Followingadjustment of the pH to 3.0 to 5.5, the solution is cooledto a temperature not substantially greater than 5 C. A low molecular weight aliphatic alcohol in an amount sufficient to bring the concentration of alcohol in the solution to at least mols per liter is added to the cooled solution at such a rate thatthe temperature does not rise above 5 C. Addition is facilitated if the methanol is chilled before addition to the solution. Methanol, ethanol, and isopropanol have been found particularly suitable. Albumin is precipitated upon the addition of alcohol and the combined precipitate of alphaglobulins and albumin is removed from solution by appr priate means such as centrifugation. It is essential that 'alpha-globulins be precipitated before alcohol is added to the solution and that alpha-globulins in solution net comein contact with alcohol or they will be denatured. The temperature of a solution containing albumin must not be substantially above 5 C. when alcohol is added in order that there be no 'denaturation of the albumin. Upon adjustment of the pH to 6.5 to 7.5, an aqueous solution of the precipitate containing alpha-globulins and albumin is suitable for use in the Diamond test. If dc sired,.the precipitate. may be dissolved in distilled water and dialyzed to remove salts and alcohol. The invention is further illustrated by the following example butit should be understood that the invention is not to be lim- 1 ited to the specificdetails set forth in the example.

normal hydrochloric acid..- The solution was heated for thirty minutes at a temperature of 75 C. while being continually stirred mechanically and thencooledtoroom temperature and adjusted to a pH of 5.6 with hydrochloric acid, The precipitate of denatured beta-globulins formed at this point were removed by filtration and the clear liquid was adjusted to a pH of 4.5 with hydrochloric acid.

After cooling'the solution to 0.59 (3., 2334 cc. of methanol, previously. cooled to 5 C. were added at such a rate 'that the temperature remained between 0 7 and 5 C. Precipitated alpha-globulinsand albumin were removed from the solution by centrifugation and during the time of removal the temperature of the solution was: maintained at -5 C, The alcohol associated with the precipitate removed by centrifugationwas removed under vacuum. The precipitate was dissolved in 125 cc. of

0.1%saline and adjusted f'withi sodium hydroxide to a pH 7 of" 7.2.. I J p The following example illustrates the use of the saline solution of alpha-globulins and albumin prepared ac-,

cording to the above procedurein the" test procedure of Diamond, the said saline solution'being'isubstitutedlfor the solution of human or bovine, albumin, acacia, globi n or other macromolecular substance used in the Diamond procedure.

A-blood sample suspected of containing the'blocking 7 antibody was allowed to clot and the clotted sample was centrifuged. Two drops of'the serum obtained by centrifugation were placed in a test tube and to this was added one drop of the physiological salt solution'obtained by the above procedure which contained in solution 22% tained at 37 C., removed .from the water bath; can trifuged at 1000 revolutions per minute for one, minute,.

by weight of the blood fraction'comprising alpha-globulins' and albumim two per cent, by weight of Rh positivejf blood cells were suspended in the mixture. Theimix ture was incubated for one hour in a water bath mainremoved from the centrifuge, agitated gently and exam! ined microscopically. The red blood cells enhibited'the phenomenon known as clumping, thus demonstrating the presence in the test serum of the blocking antibody.

Since certain changes'may be made in the above process and diiferent embodiments-of the invention could be able for use in demonstrating incomplete Rh antibodies by suspending reacting blood cells in a solution of the blood serum proteins which comprises: adding to mammalian blood serum an alkali metal salt of an organic acid selected from the group consisting of fatty acids having from 4 to 10 carbon atoms and acetyltryptophane in an amount sufiicient to bring the molarityof'the s'erum' with respect thereto to within the range of 0.13-0.17; adjusting the pH of the solution to 6.8-8.2; heating the solution; cooling the solution; adjusting the pH of the solution to 5.55-5.60, whereby a precipitate of denatured globulins is formed; removing the precipitated denatured globulins from the solution; adjusting the pH of the solution to 3.0-5.5, whereby alpha-globulins are precipitated; cooling the solution to a temperature not substantially above 5 C.; adding a low molecular weight aliphatic alcohol to the solution in an amount suflicient to bring the concentration of alcohol in the solution to at least mols per liter and at such a rate that the temperature of the solution does not rise above 5 C. during the course of the addition, whereby albumin is precipitated; and removing precipitated alpha-globulins and albumin from the solution.

2. The process for preparing blood serum proteins suit able for use in demonstrating incomplete Rh antibodies by suspending reacting blood cells in a solution of the blood serum proteins which comprises: adding to mammalian blood serum an alkali metal salt of an organic acid, selected from the group consisting of fatty acids having from 4 to 10 carbon atoms and acetyl'tryptophane, in an amount suflicient to bring the molarity of the serum with respect thereto to within the range of 0.13-0.17; adjusting the pH of the solution to 7.0-8.0; heating the solution to a temperature within the range of 70 75 C. and maintaining the solution at this temperature for 25- 35 minutes; cooling the solution to approximately room temperature; adjusting the pH of the solution to 5.55-

5.60; whereby a precipitate of denatured globulins is formed; removing the precipitated denatured globulins from the solution; adjusting the pH of the solution to 3.3-4.8 whereby alpha-globulins are precipitated; cooling the solution to a temperature not substantially greater than 5 0.; adding methanol to the solution in an amount sufiicient to bring the concentration thereof in the solution to at least 10 mols per liter and at such a rate that the temperature of the solution does not rise above 5 C. during the course of the addition, whereby albumin is precipitated; and removing precipitated alpha-globulins and albumin from the solution.

3. A process according to claim 1 in which the alkaliv metal salt of an organic acid is sodium capryllate.

4. A process according to claim 2 in which the alkali metal salt of an organic acid is sodium capryllate.

References Cited in the file of this patent McCulloch: Nature, vol. 165, pp. 276-277, February 18, 1950.

Reid et al.: Am. J. Clin. PathoL, vol. 19, No. 1, pp. 10-15, January 1949.

Greenberg: Amino Acids and Proteins, 1st ed., 1951, pp. 536, 727, 282 relied upon.

Cohn et al.: I. A. C. S., vol. 68, March 1946, pp. 459- 475 (p. 460 relied upon).

Liu et al.: Chinese J. Physiol., Vol. 8, No. 2, 1934, pp. 97-109 (pp. 97, 98, 99, 100, 101 and 109 relied upon). 

1. THE PROCESS OF PREPARING BLOOD SERUM PROTEINS SUITABLE FOR USE IN DEMONSTRATING INCOMPLETE RH ANTIBODIES BY SUSPENDING REACTING BLOOD CELLS IN A SOLUTION OF THE BLOOD SERUM PROTEINS WHICH COMPRISES: ADDING TO MAMMALIAN BLOOD SERUM AN ALKALI METAL SALT OF AN ORGANIC ACID SELECTED FROM THE GROUP CONSISTING OF FATTY ACIDS HAVING FROM 4 TO 10 CARBON ATOMS AND ACETYLTRYPTOPHANE IN AN AMOUNT SUFFICIENT TO BRING THE MOLARITY OF THE SERUM WITH RESPECT THERETO TO WITHIN THE RANGE OF 0.13-0.17; ADJUSTING THE PH OF THE SOLUTION TO 6.8-8.2; HEATING THE SOLUTION; COOLING THE SOLUTION; ADJUSTING THE PH OF THE SOLUTION TO 5.55-5.60, WHEREBY A PRECIPITATE OF DENATURED GLOBULINS IS FORMED; REMOVING THE PRECIPITATED DENATURED GLOBULINS FROM THE SOLUTION; ADJUSTING THE PH OF THE SOLUTION TO 3.0-5.5, WHEREBY ALPHA-GLOBULINS ARE PRECIPITATED; COOLING THE SOLUTION TO A TEMPERATURE NOT SUBSTANTIALLY ABOVE 5* C.; ADDING A LOW TEMPERATURE NOT SUBSTANTIALLY ALCOHOL TO THE SOLUTION IN AN AMOUNT SUFFICIENT TO BRING THE CONCENTRATION OF ALCOHOL IN THE SOLUTION TO AT LEAST 10 MOLS PER LITER AND AT SUCH A RATE THAT THE TEMPERATURE OF THE SOLUTION DOES NOT RISE ABOVE 5* C. DURING THE COURTSE OF THE ADDITION, WHEREBY ALBUMIN IS PRECIPITATED; AND REMOVING PRECIPITATED ALPHA-GLOBULINS AND ALBUMIN FROM THE SOLUTION. 